Yeast Transformations

The goal of my project is to determine if the evolution of sexual agglutinin genes is a possible mechanism behind speciation. These past two weeks, I’ve been performing transformations to insert the sexual agglutinin genes I am analyzing into European, Migrant and North American strains of Saccharomyces paradoxus.

My first step in performing a transformation involves Polymerase Chain Reaction (PCR). PCR amplifies the segment of DNA containing the sexual agglutinin gene of interest. There are three main steps in PCR. First, DNA strands are separated by heating the environment to very high temperatures. Next, the temperature is lowered, allowing primers, short strands of DNA that mark starting points for DNA synthesis, to attach to the single stranded DNA molecules. Finally, DNA polymerase, an enzyme that replicates DNA, binds to the primers and forms the complementary strand, replicating the gene of interest. After PCR, I test to see whether the process was successful using gel electrophoresis. In this technique, molecules are separated based on their size and charge. I load my samples in the wells at the top of the gel and apply an electric current. One end of the gel box is connected to a positive electrode while the other end is connected to a negative electrode. DNA is negatively charged, so DNA molecules travel towards the positive electrode. Shorter fragments of DNA run through the gel faster than larger ones. Once gel electrophoresis is finished, I examine the bands formed from the fragments of DNA. By comparing the bands in my samples to a ladder, I determine whether the gene of interest was amplified or not.

After performing PCR, I can begin my transformation. During a transformation, I make the yeast cell walls porous so that I can insert the gene of interest from my PCR product into their genomes using single stranded DNA as a carrier. Next I plate the yeast onto antibiotic plates. The DNA from my PCR product contains an antibiotic resistance gene that flanks the gene of interest, so if a yeast cell grows on an antibiotic plate, I know it must contain the gene of interest.

Next, I place the yeast on a low nutrient medium, simulating each yeast cell to undergo sporulation. Sporulation results in a tetrad of four haploid spores. Two of the spores contain the sexual agglutinin gene of interest while the other two do not. I dissect, or separate, the spores and determine which ones possess the gene of interest.

I’ve really enjoyed working in the lab this summer. I have begun my dissections, though I am still learning the proper technique. It’s still difficult for me to separate the yeast asci. Hopefully, my dissections will improve as I continue to practice, and I will be able to move on to the next steps of my project soon!

 

Comments

  1. anwesterhaus says:

    I love how thorough and easy to understand your description of your experimental procedure is! I wish you luck with your project and the next steps! I was wondering if you could clarify how inserting the agglutinin gene into the different strains of yeast will help determine whether evolution of sexual agglutinin genes is a mechanism behind speciation?

  2. Hi! Thank you for your comments! I’d be glad to try and clarify.
    In a previous project, my lab partner and I computationally determined the rates of evolution for different families of genes in Saccharomyces paradoxus. We found that the sexual agglutinin family of genes has higher rates of evolution compared to other families of genes. Since sexual agglutinin genes are required for yeast to mate and reproduce, and because speciation is considered complete when the newly formed species is no longer able to reproduce with members of its ancestral species, I think if the alleles are swapped among various strains, their mating behaviors will change. I am specifically interested in seeing if giving a Migrant a European sexual agglutinin allele will result in the loss of mating discrimination towards North American potential mates. If this does occur, then the evolution of the sexual agglutinin allele could be a possible mechanism behind speciation, and if it does not then there must be some other mechanism.
    I hope this clarifies the thinking behind my project!

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